Systematic Analysis of Human Telomeric Dysfunction Using Inducible Telosome/Shelterin CRISPR/Cas9 Knockout Cells

OPEN Citation: Cell Discovery (2017) 3, 17034; doi:10.1038/celldisc.2017.34 ARTICLE www.nature.com/celldisc Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells Hyeung Kim1, Feng Li2, Quanyuan He1, Tingting Deng2, Jun Xu3, Feng Jin4, Cristian Coarfa4, Nagireddy Putluri4, Dan Liu1,3, Zhou Songyang1,2,* 1Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA; 2Key Laboratory of Gene Engineering of the Ministry of Education and State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, China; 3Cell-Based Assay Screening Service Core, Baylor College of Med- icine, Houston, TX, USA; 4Department of Molecular and Cellular Biology and Advanced Technology Core, Baylor College of Medicine, Houston, TX, USA CRISPR/Cas9 technology enables efficient loss-of-function analysis of human genes using somatic cells. Studies of essential genes, however, require conditional knockout (KO) cells. Here, we describe the generation of inducible CRISPR KO human cell lines for the subunits of the telosome/shelterin complex, TRF1, TRF2, RAP1, TIN2, TPP1 and POT1, which directly interact with telomeres or can bind to telomeres through association with other subunits. Homozygous inactivation of several subunits is lethal in mice, and most loss-of-function studies of human telomere regulators have relied on RNA interference-mediated gene knockdown, which suffers its own limitations. Our inducible CRISPR approach has allowed us to more expediently obtain large numbers of KO cells in which essential telomere regulators have been inac- tivated for biochemical and molecular studies. Our systematic analysis revealed functional differences between human and mouse telomeric proteins in DNA damage responses, telomere length and metabolic control, providing new insights into how human telomeres are maintained. Keywords: CRISPR/Cas9; inducible knockout; metabolism; POT1 isoform; telomere; telosome/shelterin Cell Discovery (2017) 3, 17034; doi:10.1038/celldisc.2017.34; published online 26 September 2017 Introduction instance, TRF1 and TRF2 bind directly to the telomere duplex through their myb domains [9–13], whereas In the past 20 years, we have gained tremendous POT1 binds 3’ single-stranded (ss) telomeric overhangs insight into how the ends of mammalian chromosomes [14, 15]. RAP1 is recruited by TRF2, but apparently does or telomeres are maintained and regulated. Together not directly interact with any of the other subunits [16]. with the telomerase, which consists of the reverse tran- TIN2 can interact with both TRF1 and TRF2 [6, 17–21]. scriptase TERT and RNA template TR/TERC, a mul- It also binds TPP1 and helps bring to telomeres the titude of telomere-binding proteins participate in TPP1-POT1 heterodimer that is essential for regulating – telomere maintenance [1 5]. In particular, six core telo- telomerase access to telomeres [21–30]. The core telomeric proteins, TRF1, TRF2, RAP1, TPP1, TIN2 and mere proteins often act as interaction hubs to recruit POT1, dynamically assemble on telomeres as a large factors of diverse pathways to telomeres and ensure complex called telosome or shelterin and are essential in crosstalk between telomere maintenance pathways and telomere length regulation and end protection in mam- other cellular processes [8, 19, 31, 32]. In fact, several key – mals [6 8]. Extensive research has revealed the interac- telomere regulators have been shown to regulate meta- tions and functions of telosome components. For bolism, providing direct evidence of the close ties between telomere regulation and metabolic control. For *Correspondence: Zhou Songyang example, the human telomerase reverse transcriptase has Tel: +713 798 5220; Fax: +713 796 9438 E-mail: [email protected] been found to localize to the mitochondria and reduce Received 15 March 2017; accepted 27 July 2017 intracellular oxidative stress [33–36]. Our lab has found Studying telosome subunits using inducible knockout cell lines 2 that TIN2 can also localize to the mitochondria and of their human orthologs, we first turned to RNAi KD regulate oxidative phosphorylation [37]. in human cells through stable expression of short Numerous studies have demonstrated that dysfunc- hairpin RNAs (Supplementary Figure S1A). Even with tional telomeres can lead to telomere length defects, effective KD (480%) of TRF2, for example, we could deprotected telomeres, genomic instability and diseases only observe minor DNA damage responses (DDRs) [1, 4, 32, 38]. Much of our knowledge regarding the at telomeres (data not shown), rarely more severe molecular and functional significance of mammalian telo- phenotypes such as chromosome end-to-end fusions meric proteins comes from studies using mouse knockout found in Trf2 KO MEF cells [50], suggesting that (KO) mouse embryonic fibroblast (MEF) cells, as genes residual TRF2 proteins in the KD cells may have been are more readily targeted in mouse embryonic stem cells. sufficient to prevent severe and sustained telomere However, notable differences exist in telomere regulation DNA damages. We next attempted straight KO of between mouse and human. For instance, human telo- these genes by CRISPR/Cas9, but failed to isolate any meres are considerably shorter than those of laboratory clones of TRF2, TIN2 or POT1 KO cells. Given such mice and human has one POT1 gene, whereas mouse has findings, we decided that human cells conditionally two (Pot1a and Pot1b). Such disparities underscore the knocked out for telosome subunits would be more need for loss-of-function human cellular models. Majority desirable. of the loss-of-function studies in human cells have relied on Traditionally, conditional KO alleles are generated RNA interference (RNAi)-mediated inhibition of endo- by inserting into some particular locus recombination genous genes. The limitations of RNAi knockdown (KD) sequences such as loxp sites, which can mediate the and the fact that several key telomere regulators including deletion of intervening sequences upon the expression TRF2 and TIN2 are essential genes have complicated data of recombinases such as Cre [51–53]. The insertion of analysis and interpretation. Complete inactivation of these such exogenous sequences may alter gene regulation telomere regulatory genes in cells may cause cell death, and the entire process is often time consuming. To precluding further detailed biochemical and molecular generate conditional telosome subunit KO cells, we studies, especially experiments that require extended cul- modified a lentivirus-based inducible CRISPR/Cas9 turing and/or large numbers of cells. KO system [54], and constructed separate vectors for The advent of the CRISPR/Cas9 genome-editing tech- inducible Cas9 and constitutive single guide RNA nology has afforded investigators unprecedented opportu- (sgRNA) expression (Supplementary Figure S1B). nities to more efficiently and specifically target genes in Hela cells were transduced with lentiviruses encoding human cells and to explore the consequences of their inducible Cas9 and a clone in which Cas9 expression inactivation [39–47]. In this study, we took advantage of the could be reproducibly activated with doxycycline in a highly flexible and adaptable CRISPR/Cas9 system and dose-dependent manner was selected (Supplementary generated human inducible KO cell lines for each of the Figure S1C). telosome components. This panel of cells has allowed us to The double-strand breaks resulting from Cas9 clea- survey the functional significance of each telomeric protein vage trigger the non-homologous end joining DNA and probe the impact of individual subunit inhibition on repair pathway in the absence of a donor template telomere regulation as well as metabolic control. With this [55–57]. Non-homologous end joining-mediated DNA systematic analysis of the function of human telomere repair may generate small insertions and/or deletions proteins using inducible KO cell lines, we are able to better (indels) at the target site, and compromise gene func- delineate the differences between mouse and human telo- tion if cleavage occurs within protein coding sequences. mere biology. In addition, our panel of inducible KO cell Repair of a single Cas9 cleavage site has a 1/3 chance of lines should prove invaluable to investigators seeking to in-frame ligation of the coding sequences, which may further explore the consequences of telomere dysfunction not completely disrupt gene function. We reasoned that and to study how diverse cellular functions may be dis- simultaneous targeting with two sgRNAs should rupted upon telomere dysregulation. improve the odds of larger deletions and more complete inhibition of endogenous genes. To test this Results strategy, the inducible Cas9 cells were infected with two viruses encoding two separate TIN2-specific sgRNAs Using CRISPR/Cas9 to generate inducible KO human either singly or together, selected with appropriate cell lines antibiotics, and then cultured in doxycycline- Trf1, Trf2 and Tin2 have been reported to be containing media to induce Cas9 expression essential genes in mouse [48–50]. To determine the roles (Figure 1a). At different time points following Cell Discovery | www.nature.com/celldisc Hyeung Kim et al. 3 cell lines showed similar growth patterns in the absence of doxycycline, differences in growth rates became apparent between doxycycline-induced TIN2 KO cells after 4-day treatment. Growth of the single sgRNA TIN2 KO cells

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